Process for producing low endotoxin chitosan

ABSTRACT

The present invention relates to a process for producing a low endotoxin alkali chitosan, chitin, chitosan derivative or chitin derivative, and also to a process for producing low endotoxin neutral chitosan, chitosan salt and chitosan derivatives, and to the products of such processes. The process comprises contacting chitosan, chitin, chitosan derivative or chitin derivative with an alkali solution having a concentration of less than 0.25M to form a mixture; leaving the mixture for a period of less than 12 hours and optionally drying the mixture. The low endotoxin alkali chitosan may be used in the manufacture of other useful chitosan based products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase entry of PCT/GB2014/051646,with an international filing date of May 29, 2014, which claims priorityto and the benefit of GB 1309607.8, filed on May 29, 2013, the contentsof which are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a process for producing a low endotoxinalkali chitosan, and also to a process for producing low endotoxinneutral chitosan, chitosan salt and chitosan derivatives, and to theproducts of such processes.

BACKGROUND

Chitosan is particularly useful in the preparation of haemostaticmaterials for use in controlling bleeding.

Chitosan is a derivative of solid waste from shell fish processing andcan be extracted from fungus culture. Chitosan is a water insolublecationic polymeric material. Before using chitosan in haemostaticmaterials, it is often first converted into a water soluble salt. Thisway, the chitosan salt is soluble in blood to form a gel which stemsblood flow.

Chitosan salts are ideally suited for the applications described hereinas chitosan is readily broken down in the body. Chitosan is converted toglucosamine by the enzyme lysozyme and is therefore excreted from thebody naturally. It is not necessary to remove chitosan from the body.Furthermore, chitosan salts exhibit mild antibacterial properties and assuch their use reduces the risk of infection.

In order to utilise chitosan in the preparation of haemostatic materialsthat are suitable for use in controlling bleeding, it is necessary toensure that the chitosan has a sufficiently low concentration ofendotoxin.

Endotoxin is a lipopolysaccharide existing on the surface of the outermembrane of gram-negative bacteria. Endotoxins are highly toxic tomammals, particularly humans, and are notoriously difficult to removefrom materials. Endotoxins may become pyrogenic when released into thebloodstream or other tissue where they are not usually found. Thus,endotoxin must be removed from pharmaceutically acceptable products.

Treatments to remove or destroy pyrogens, particularly endotoxin, arereferred to as methods of ‘depyrogenation’. Techniques for thedepyrogenation of materials containing endotoxin include ion exchangechromatography, ultrafiltration, distillation and various chemicalprocesses aimed at destroying endotoxin.

WO2008063503 relates to a method of removing endotoxin from chitosanincluding the following steps:

-   -   a) utilizing sterile pyrogen-free equipment and materials in a        sterile environment;    -   b) swelling chitosan containing endotoxins for up to 24 hours;    -   c) dissolving 1 kg/25 L to 1.5 kg/25 L of the chitosan in 0.01M        to 4.0M of a hydroxide base;    -   d) continuously stirring the resulting chitosan base solution;    -   e) heating the solution between 60-100° C. for 45 minutes to 4        hours with stirring;    -   f) rinsing the solution with up to 10× volume of ultra-pure        endotoxin-free water;    -   g) neutralizing the solution to a pH between 6.8 and 7.5;    -   h) forming an ultra-pure low endotoxin chitosan slurry and        transferring to a endotoxin-free closed system;    -   i) removing excess water from the slurry.

This is a complicated and costly process, especially with the need forsterile equipment and the need to rinse the solution with 10× volume ofendotoxin-free water.

US2006293509 relates to a method of making a water soluble chitosanhaving low endotoxin by:

-   -   (a) contacting water-insoluble chitosan with a basic solution        for a first period of time of greater than 1 hour;    -   (b) rinsing the water-insoluble chitosan to remove residual        basic solution, desirably with endotoxin-free water;    -   (c) partially acetylating the water-insoluble chitosan in a        reaction solution containing a phase transfer agent;    -   (d) dissolving the partially acetylated water-soluble chitosan        in an aqueous solution containing a surfactant and having a pH        of from about 7.0 at about 7.4;    -   (e) adding a water-miscible solvent into the aqueous solution        and further adjusting the pH of the aqueous solution to a pH of        at least 8.0 to cause precipitation of water-soluble chitosan        having low endotoxin content from the aqueous        solution/water-miscible solvent mixture; and    -   (f) optionally washing in a non-solvent such as isopropanol.

However, this process is complicated and expensive and desirablyinvolves using large quantities of endotoxin-free water or otherliquids. The process also requires the use of phase transfer agents andtakes place over a few hours.

TW593342 relates to a method of reducing endotoxin in chitosan by:

-   -   (a) dissolving chitosan containing endotoxin in an aqueous        solution;    -   (b) contacting the aqueous solution with a surfactant to form an        insoluble solid and an aqueous solution reduced in the content        of the endotoxin;    -   (c) using a solid/liquid separation means to separate the solid        from the aqueous solution.

However, this process requires a surfactant to react with the dissolvedchitosan to make an insoluble solid. The resulting solid will be amixture of chitosan and surfactant or a reaction product between thechitosan and surfactant.

The present invention aims to alleviate the aforementioned difficulties.

SUMMARY

According to a first aspect of the present invention, there is provideda process for producing a low endotoxin alkali chitosan, chitin or aderivative thereof, the process comprising the steps of:

-   -   (a) contacting chitosan, chitin, a chitosan derivative or a        chitin derivative with an alkali solution having a concentration        of less than 0.25M to form a mixture; and    -   (b) leaving the mixture for a period of less than 12 hours.

The process of the present invention may further comprise the step (c)of drying the mixture.

The process of the present invention provides an effective way ofobtaining an alkali chitosan, chitin, chitosan derivative or chitinderivative having a low endotoxin concentration. Advantageously, theprocess does not require a washing step, a rinsing step, use of asurfactant or phase transfer agents, sterile equipment and/or the use ofendotoxin free water. Further, specialist air filtration or sterileconditions are also not required. The process of the present inventionpreferably does not comprise a step of acetylating the chitosan.Further, the process of the present invention does not use endotoxinfree equipment. This is particularly beneficial as it reduces the costof the process compared to processes requiring such equipment.

By the term ‘chitosan derivative’ it is meant herein a partiallydeacetylated chitin, which may have different percentages ofdeacetylation, as desired. Typically, the partially deacetylated chitinsuitable for use in the present invention has a deacetylation degreeabove about 50%, more typically above about 75% and most typically 5above about 85%.

Also herein included within the term ‘derivatives’ are reaction productsof chitosan or chitin with other compounds. Such reaction productsinclude, but are not limited to, carboxymethyl chitosan, hydroxyl butylchitin, N-acyl chitosan, O-acyl chitosan, N-alkyl chitosan, O-alkylchitosan, N-alkylidene chitosan, O-sulfonyl chitosan, sulfated chitosan,phosphorylated chitosan, nitrated chitosan, alkalichitin,alkalichitosan, or metal chelates with chitosan, etc.

Whilst the first aspect of the present invention provides a process forproducing low endotoxin chitosan, chitin or a derivative thereof, it isdescribed hereinafter in relation to chitosan for convenience andillustrative purposes only.

The chitosan may be commercially available chitosan, such as food grade,medical grade or pharmaceutical grade chitosan. The process of thepresent invention may therefore be operable to provide low endotoxinalkali chitosan from commercially available chitosan. This is differentto certain processes where endotoxins may be removed or reduced as partof a chitosan production process. Beneficially, the process of thepresent invention can be used to provide low endotoxin alkali chitosanfrom prepared chitosan that would otherwise have been unsuitable to themedical field due to its endotoxin concentration.

The term alkali chitosan is used herein to refer to a chitosancomposition having a pH value of greater than pH 7.5.

The term alkali solution is used herein to refer to a solution having apH value of greater than pH 7.5.

Since the molecular weight of endotoxins can vary significantly,endotoxin concentration is measured in endotoxin units (EU) per gram ofmaterial. The measurement of endotoxin concentration is a quantificationof endotoxin levels relative to a specific quantity of referenceendotoxin.

For example, in the present invention, the concentration of endotoxin ismeasured in endotoxin units (EU) per gram of chitosan. The term ‘lowendotoxin’ is used herein to refer to an endotoxin concentration of lessthan 50 endotoxin units (EU) per gram of chitosan.

The process of the present invention is thus suitable for making analkali chitosan that has an endotoxin concentration of less than 50EU/g.

Preferably, the resulting alkali chitosan has an endotoxin concentrationof less than 30 EU/g, more preferably less than 20 EU/g, more preferablyless than 15 EU/g, even more preferably less than 10 EU/g and mostpreferably less than 5 EU/g.

It has been found that low concentrations of alkali solution produce aproduct with desirable properties. The concentration of alkali solutionused in the process may be 0.2M or less. Preferably, the concentrationof alkali solution is from around 0.01M to 0.2M. More preferably, theconcentration of alkali solution is from around 0.02M to 0.1M. Theconcentration of alkali solution may be around 0.04M to 0.06M, typically0.05M. Concentrations of alkali solution can be up to around 0.01M,0.05M, 0.10M, 0.15M, 0.20M or 0.25M. Good results have been observedwith a concentration of 0.1M alkali solution.

In some embodiments, the quantity of alkali solution to chitosan may bein the range of from about 1 part chitosan to about 10 parts alkalisolution up to about 10 parts chitosan to about 1 part alkali solution.Preferably, the quantity of alkali solution to chitosan is about 1 partalkali solution to about 2 parts chitosan, more preferably about 1 partalkali solution to about 1 part chitosan.

The alkali solution may comprise an alkali or alkaline earth componentselected from the following, either alone or in combination: metalhydroxides, metal carbonates, metal bisulphites, metal persilicates,conjugate bases and ammonium hydroxide.

Suitable metals include sodium, potassium, calcium, or magnesium.

Preferably, the alkali component is sodium hydroxide, potassiumhydroxide or sodium carbonate. Typically, sodium hydroxide is used.

The alkali solution may be contacted with the chitosan by any suitablemeans known in the art. For example, the alkali solution may be sprayedonto the chitosan or the chitosan may be mixed with the alkali solution.Preferably, there is an even distribution of alkali contacted chitosan.

Preferably, the chitosan is mixed with the alkali solution. At lowmolecular weights, the chitosan may completely or partially dissolve inthe alkali solution. The chitosan may be mixed with the alkali solutionin step (a) for up to around 30 minutes, more preferably for around 10minutes. In some embodiments, the chitosan may be mixed with the alkalisolution for greater than 30 minutes.

In some embodiments, the chitosan does not dissolve in the alkalisolution.

In some embodiments, the chitosan does not swell in the alkali solution.

In some embodiments, the alkali solution wets the chitosan withoutdissolving or swelling the chitosan.

In some embodiments, the mixture of chitosan and alkali solution may bestirred intermittently for the duration of step (b).

The mixture of chitosan and alkali solution is left for a period of timein which sufficient endotoxin is destroyed. The mixture of chitosan andalkali solution is left for a period of less than 12 hours. It has beendiscovered that leaving the chitosan and alkali solution having aconcentration of less than 0.25M for a period of time of less than 12hours before subsequent processing results in a desirably low endotoxinconcentration in the resulting alkali chitosan.

It is an advantage of the process of the present invention that themixture can be left without the need for continued mixing of thechitosan with the alkali solution.

In some embodiments, the mixture may be left in step (b) for a period ofless than 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 hours. The mixture may beleft in step (b) for a period of less than 10 hours, preferably lessthan 8 hours, more preferably less than 6 hours, even more preferablyless than 4 hours and most preferably less than 2 hours.

In some embodiments, the mixture may be left in step (b) for a period ofmore than 1 but less than 12 hours; more than 2 but less than 12 hours;more than 3 but less than 12 hours; more than 4 but less than 12 hours;more than 5 but less than 12 hours; more than 6 but less than 12 hours;more than 7 but less than 12 hours; more than 8 but less than 12 hours;more than 9 but less than 12 hours; more than 10 but less than 12 hours;or more than 11 but less than 12 hours. In some embodiments, the mixturemay be left in step (b) for a period of between 1 to 11 hours, 1 to 10hours, 1 to 9 hours, 1 to 8 hours, 1 to 7 hours, 1 to 6 hours, 1 to 5hours, 1 to 4 hours, 1 to 3 hours or 1 to 2 hours. Thus, in someembodiments, the mixture may be left for a period of between 2 to 10hours, 4 to 8 hours or 5 to 7 hours.

In some embodiments, the mixture may be left in step (b) for a period ofless than 1 hour, including less than 60, 55, 50, 45, 40, 35, 30, 25,20, 15, 10, or 5 minutes. The mixture may be left for a period of lessthan three minutes, less than two minutes or less than one minute.

In some embodiments, the mixture may be left in step (b) only for theperiod of time taken to prepare the mixture for a subsequent stage ofprocessing, for example, the drying step (c). Good results have beenobserved when the mixture has been dried immediately followingcontacting chitosan with the alkali solution in step (a). In thiscontext, immediately means that the mixture is only left in step (b) forthe period of time it takes to prepare the mixture for the drying step(c). Typically, this is less than about 30 seconds, preferably less than20 seconds and most preferably less than 10 seconds.

Thus, according to an aspect of the present invention there is provideda process for producing a low endotoxin alkali chitosan, chitin or aderivative thereof, the process comprising the steps of:

-   -   (a) contacting chitosan, chitin, a chitosan derivative or a        chitin derivative with an alkali solution having a concentration        of less than 0.25M to form a mixture; and    -   (b) immediately drying the mixture.

In such a process, the mixture is left in step (b) only for the time ittakes to prepare it for the next stage of processing. For example, themixture may be left in step (b) for the time it takes to prepare it fordrying. The mixture may then be dried in a drying step (c).

The mixture may be left to stand in step (b) at room temperature andpressure. By room temperature and pressure, it is meant a temperature ofaround 20-25° C. and a pressure of about 1 atmosphere (atm).Beneficially, the mixture does not need to be left in a sterileenvironment.

The mixture is preferably stored in a clean container. The mixture maybe stored under an inert atmosphere.

The mixture may further comprise a preservative. Beneficially, thepreservative may eliminate the risk of microbial growth that maydevelop, for example, when the mixture is left for a prolonged period.The preservative may be any preservative that is biocompatible andsuitable for use in an alkali environment. Suitable preservativesinclude silver ions, zinc ions, chlorohexadine, or combinations thereof.

The process of the present invention may or may not comprise the dryingstep. The drying step may be performed by any conventional drying meansknown in the art. Preferably, the drying step is performed in an oven orby filtration through an air dryer. Again, specialist sterile equipmentis not required for the drying step.

It has been discovered that, once the mixture has been dried in thedrying step, the endotoxin level of the dry mixture does not noticeablyincrease over time. This is beneficial in that the mixture can be storedfor a period of time prior to further processing.

There is thus provided a low endotoxin alkali chitosan having anendotoxin concentration of less than 50 EU/g. The low endotoxin alkalichitosan may be water insoluble. At low molecular weights, the lowendotoxin alkali chitosan may show some water solubility.

According to a further aspect of the present invention, there isprovided a low endotoxin alkali chitosan, chitin or a derivative thereofobtainable by the process as described herein.

According to a further aspect of the present invention, there isprovided an alkali chitosan, chitin or a derivative thereof comprisingan endotoxin concentration of less than 50 EU/g.

The alkali chitosan, chitin or a derivative thereof preferably has anendotoxin concentration of less than 30 EU/g, preferably less than 20EU/g, more preferably less than 15 EU/g, even more preferably less than10 EU/g and most preferably less than 5 EU/g.

The low endotoxin alkali chitosan, chitin or a derivative thereofcomprises alkali having a concentration of less than 0.25M. Preferably,the concentration is around 0.2M or less, more preferably from around0.15M or less and even more preferably from around 0.1M or less.

The low endotoxin alkali chitosan may be used as an intermediate in themanufacture of other chitosan products, such as for example, derivativesor copolymers or in the manufacture of low molecular weight chitosan orchitosan oligosaccharides. The low endotoxin alkali chitosan may also beuseful as a raw material for the manufacture of other forms of chitosanor derivatives or copolymers, such as chitosan based fibres, fabrics,coatings, films, gels, solutions, sheets or foams.

In particular, the low endotoxin alkali chitosan may be used in thepreparation of other useful chitosan products having low concentrationsof endotoxin, including neutral chitosan and chitosan salts and otherchitosan derivatives, for example, carboxymethyl chitosan, hydroxyethylchitosan, acyl chitosan, alkyl chitosan, sulphonyl chitosan,phosphorylated chitosan, alkylidene chitosan, metal chelates, chitosanchloride, chitosan lactate, chitosan acetate, chitosan malate, chitosangluconate.

Thus, according to a further aspect of the present invention there isprovided a process for producing a low endotoxin neutral chitosan,chitosan salt or chitosan derivative comprising the step of contactingan alkali chitosan prepared by the process described hereinbefore withan acid.

The process can provide medically useful neutral chitosan, chitosan saltor other chitosan derivative having low concentrations of endotoxin.

The step of contacting the alkali chitosan with the acid may beperformed before the drying step (c) described hereinabove in theprocess for producing a low endotoxin alkali chitosan.

Alternatively, the step of contacting the alkali chitosan with an acidmay be performed after the drying step (c) described hereinabove in theprocess for producing a low endotoxin alkali chitosan. In suchembodiments, the process for producing a low endotoxin neutral chitosan,chitosan salt or chitosan derivative may comprise a further drying stepafter the step of contacting the alkali chitosan with an acid. Thedrying step may be performed by any conventional drying means known inthe art. Preferably, the drying step is performed in an oven or byfiltration of the product through an air dryer.

The acid may be contacted with the alkali chitosan by any suitable meansknown in the art. For example, the acid may be sprayed onto the alkalichitosan or the alkali chitosan may be mixed with the acid.

Preferably, the alkali chitosan is mixed with the acid.

A neutral chitosan is referred to herein to mean a chitosan compositionhaving a pH value of between about pH 6.5 and about pH 7.5, andpreferably about pH 7.

Thus, in order to prepare a neutral chitosan, the alkali chitosan may bemixed with an appropriate volume and/or concentration of acid to form aneutral solution having a pH of between 6.5 and 7.5. The volume and/orconcentration of acid required to neutralise the alkali chitosan will bedependent on the pH of the alkali chitosan.

Alternatively, in order to prepare a chitosan salt or chitosanderivative, the alkali chitosan may be mixed with a volume and/orconcentration of acid in excess of that required to provide a neutralchitosan.

A suitable acid for use in the present invention may be selected fromthe following, either alone or in combination: organic acids, carboxylicacids, fatty acids, amino acids, lewis acids, monoprotic acids, diproticacids, polyprotic acids, nucleic acids and mineral acids.

Suitable organic acids may be selected from the following, either aloneor in combination: acetic acid, tartaric acid, citric acid, ascorbicacid, acetylsalicylic acid, gluconic acid and lactic acid.

Suitable fatty acids may be selected from the following, either alone orin combination: myristoleic acid, palmitoleic acid, sapienic acid, oleicacid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid,α-Linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid,docosahexaenoic acid, caprylic acid, capric acid, lauric acid, myristicacid, palmitic acid, stearic acid, arachidic acid, behenic acid,lignoceric acid, cerotic acid.

Suitable amino acids may be selected from the following, either alone orin combination: histidine, lysine, aspartic acid, glutamic acid,glutamine, glycine, proline, taurine.

Suitable mineral acids may be selected from the following, either aloneor in combination: hydrochloric acid, sulphuric acid and nitric acid.Preferably, the acid selected for the neutralisation is hydrochloricacid.

The acid may have a concentration of from about 0.001M acid up to themaximum possible concentration of acid. For example, the typical maximumconcentration for sulphuric acid is around 98% sulphuric acid. The acidmay have a concentration of from about 0.01M to 5M, 0.01M to 3M or 0.1Mto 2M. Preferably, the acid has a concentration of about 1M. Theconcentration of acid may be up to about 0.01M, 0.05M, 0.10M, 0.15M,0.20M, 0.25M, 0.30M, 0.35M, 0.40M, 0.45M, 0.50M, 0.55M, 0.60M, 0.65M,0.70M, 0.75M, 0.80M, 0.85M, 0.90M, 0.95M or 1.0M.

The acid may be present as an acid liquor comprising the acid and anon-solvent. The non-solvent may be any solvent in which chitosan isinsoluble. Typical non-solvents include ethyl lactate, ethyl acetate,methyl acetate, ethanol, acetone or mixtures thereof. Preferably, thenon-solvent comprises ethyl acetate or ethanol. More preferably, thenon-solvent comprises 80:20 ethanol in water. Beneficially, it has beenobserved that the reaction proceeds at a faster rate using a non-solventcomprising an 80:20 mixture of ethanol to water.

The ratio of chitosan to acid liquor may be from about 5 to 1 to about 1to 5. Preferably, the ratio of chitosan to acid liquor is about 2 to 1.

In some embodiments, the low endotoxin alkali chitosan may be mixed withthe acid for up to around 30 minutes or less, more preferably for around10 minutes or less and most preferably for around five minutes or less.The reaction may then be allowed to happen as the mixture is dried.

The product resulting from the mixture of alkali chitosan with acid maycontain an acid salt. Preferably, the alkali solution and acid areselected to ensure that the acid salt formed is biocompatible. Forexample, the alkali solution may comprise sodium hydroxide and the acidmay comprise hydrochloric acid. In such an example, the acid salt wouldbe the biocompatible salt sodium chloride.

The acid salt is formed as a by-product of the reaction between thealkali chitosan and the acid.

It has been discovered that the presence of an acid salt in the productcan affect the usefulness of the resulting chitosan product. Forexample, it has been observed that chitosan gels to a lesser extent insaline solution than it does in water, and to an even lesser extent insaline solution at double concentration. Double concentrated salinesolution referred to herein is contemplated as having an amount ofsodium chloride of 1.8%. Consequently, it is desirable to have as low anamount of acid salt in the resulting chitosan product as possible and,ideally, a level of acid salt which makes little or substantially nodifference to the effectiveness of the chitosan product.

It has surprisingly been discovered that using an alkali solution havinga low concentration, such as less than 0.25M and preferably from around0.01M to around 0.2M, produces the desired low endotoxin concentrationwhilst also resulting in less acid salt by-product being produced in thesubsequent process to produce a neutral chitosan, chitosan salt orchitosan derivative. Beneficially, less acid salt by-product has beenfound to result in a chitosan product that has improved gelling in useover products containing a higher amount of acid salt. The process ofthe present invention can provide a chitosan product with a suitably lowamount of acid salt without the need to wash or rinse the chitosanproduct. This also has the added advantage of not requiring the use ofendotoxin-free water in a washing or rinsing step.

It has also been found that using low concentrations of alkali solutionas described herein causes less of a reduction in the viscosity of thechitosan when producing a neutral chitosan, chitosan salt or chitosanderivative.

By low concentrations of alkali, it is meant less than 0.25M, preferablyfrom around 0.01M to 0.2M. In some embodiments, the alkali concentrationmay be from 0.02M to 0.1M, preferably 0.05M to 0.1M. Good results havebeen observed using an alkali concentration of around 0.1M. In someembodiments, the alkali concentration may be as mentioned hereinabove.Beneficially, therefore, using low concentrations of alkali solution inthe process is less damaging to the chitosan. It is therefore possibleto remove endotoxin from chitosan whilst causing only minimal change inviscosity. It is desirable for the viscosity of the chitosan to reduceby less than about 25% in the process, preferably by less than about 15%and more preferably by less than about 10%.

Where the process provides a low endotoxin neutral chitosan, the productis suitable for use as an intermediate in the production of otherchitosan based products. One particular use is in the production ofchitosan salts, whose absorbent properties make them desirable for usein haemostatic preparations for controlling bleeding. It is preferablethat the chitosan salts are water soluble.

Thus, in another embodiment of the present invention, a low endotoxinchitosan salt may be prepared by contacting a low endotoxin neutralchitosan produced by the process described herein with an acid.

The acid may be any acid appropriate for providing the desired chitosansalt. For example, if chitosan acetate is desired, acetic acid may beused; if chitosan succinate is desired, succinic acid may be used, etc.Any of the acids described herein may be used in the present process forproducing a low endotoxin chitosan salt.

The process for producing a low endotoxin chitosan salt or chitosanderivative may further comprise the step of drying the mixture of lowendotoxin neutral chitosan and acid. The drying step may be performed byany conventional drying means known in the art. Preferably, the dryingstep is performed in an oven or by filtration of the product through anair dryer.

There is thus provided a low endotoxin neutral chitosan, chitosan saltor chitosan derivative having an endotoxin concentration of less than 50EU/g.

The low endotoxin neutral chitosan may be water insoluble.

The low endotoxin chitosan salt may be water soluble.

According to a further aspect of the present invention, there isprovided a low endotoxin neutral chitosan, chitosan salt or chitosanderivative obtainable by any of the processes described herein.

According to a further aspect of the present invention, there isprovided a neutral chitosan, chitosan salt or chitosan derivativecomprising an endotoxin concentration of less than 50 EU/g.

The neutral chitosan, chitosan salt or chitosan derivative may have anendotoxin concentration of less than 30 EU/g, preferably less than 20EU/g, more preferably less than 15 EU/g, even more preferably less than10 EU/g, and most preferably less than 5 EU/g.

The low endotoxin chitosan salt of the present invention is suitable foruse as a haemostat for stemming blood flow.

Thus, according to a further aspect of the present invention, there isprovided a low endotoxin chitosan salt as described herein for use as ahaemostat for stemming blood flow. The low endotoxin chitosan salt canbe used as a haemostat for internal or external bleeding. For chitosansalts used in surgery for internal bleeding, endotoxin concentration ofless than 5 EU/g is desired.

The low endotoxin chitosan salt of the present invention may beincorporated into a wound dressing for superficial non-life threateningbleeding or life threatening bleeding.

Thus, according to a further aspect of the present invention, there isprovided a low endotoxin chitosan salt as described herein for use in awound dressing for superficial non-life threatening bleeding or lifethreatening bleeding.

The low endotoxin chitosan salt of the present invention is suitable foruse in the preparation of a haemostatic wound dressing for stemmingblood flow. According to a further aspect of the present invention,there is provided a haemostatic wound dressing comprising a lowendotoxin chitosan salt as described herein.

According to a still further aspect of the present invention, there isprovided a haemostatic material comprising a low endotoxin chitosan saltas described herein.

The haemostatic material and/or chitosan salt may be in any suitableform, such as particulate, powder, granular, flake, fibrous, gel, foam,sheet, film or liquid form.

According to a still further aspect of the present invention, there isprovided a method of stemming blood flow comprising the steps of:optionally cleaning a wound area where possible; applying to said woundarea a haemostatic wound dressing comprising a low endotoxin chitosansalt as described herein; and applying constant pressure to the woundarea until a gel clot forms.

Constant pressure is preferably applied to the wound area for aboutthree minutes or more.

Beneficially, the lower the concentration of alkali solution used in thepreparation of the haemostatic material of the present invention, thebetter the material performs in penetrability, blood clotting andhaemostasis.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described further inthe following non-limiting examples with reference to the accompanyingdrawing in which:

FIG. 1 is a graph displaying the effect of different concentrations ofacid salt by-product on the viscosity of a chitosan product in thedifferent media.

DETAILED DESCRIPTION

Endotoxin Testing

-   -   1. Make up USP (United States Pharmacopia) extraction solution        as detailed in USP for chitosan endotoxin testing (4.6 ml of 1M        HCl and 45.4 ml endotoxin free water);    -   2. Extract by adding 0.1 g of the test chitosan product to 9.9        ml of USP extraction solution and leave for 48 hours at 37° C.;    -   3. After 48 hours, dilute 100 μl of the extract in 0.9 ml of        endotoxin free water; and    -   4. Mix 100 μl of the above in 100 μl of Endotoxin Specific (ES)        buffer provided by Charles River.

The resulting extract is tested using an Endosafe®-PTS™ handheldspectrophotometer that utilises FDA-licensed disposable cartridges. Theextract process uses a 2000× dilution and a minimum test limit detectionof 10 EU/g.

EXAMPLES Example 1

50 g of chitosan was mixed with 50 g 0.1M NaOH for 10 mins. Theresulting wet alkali chitosan crumb was dried immediately in a fluid beddrier at 40° C.

Initial Endotoxin of raw chitosan: 64.8 EU/g

Dry treated alkali Chitosan: 16.3 EU/g

Example 2

50 g of chitosan was mixed with 50 g 0.05M NaOH for 10 mins. Theresulting wet alkali chitosan crumb was dried immediately in a fluid beddrier at 40° C.

Initial Endotoxin of raw chitosan: 64.8 EU/g

Dry treated alkali Chitosan: 20.0 EU/g

Example 3

50 g of chitosan was mixed with 50 g 0.01M NaOH for 10 mins. Theresulting wet alkali chitosan crumb was dried immediately in a fluid beddrier at 40° C.

Initial Endotoxin of raw chitosan: 64.8 EU/g

Dry treated alkali Chitosan: <30 EU/g

Example 4

50 g of chitosan was mixed with 50 g 0.1M NaOH for 8 hrs. The resultingwet alkali chitosan crumb was dried immediately in a fluid bed drier at40° C.

Initial Endotoxin of raw chitosan: 64.8 EU/g

Dry treated alkali Chitosan: 23.4 EU/g

The process can be scaled up and used to make larger batch sizes.

The process can be used on chitosan in different physical forms such asa chitosan fibre or chitosan fabric.

The process can also utilise a different base to sodium hydroxide, suchas potassium hydroxide for example.

Examples 1-4 relate to the production of low endotoxin alkali chitosan.This low endotoxin alkali chitosan can subsequently be used as a rawmaterial to make other chitosan based products. For example alkalichitosan can be neutralised to pH 7 to form a neutral chitosan by addinga low level of an appropriate acid that would react with the base tomake a biocompatible salt. For example, if sodium hydroxide is used inthe basic solution, it can be neutralised by the addition ofhydrochloric acid. The product would contain a low amount of residualsodium chloride.

The low endotoxin alkali chitosan formed in Examples 1-4 can also beused to make a low endotoxin water soluble chitosan salt or otherchitosan derivatives. Beneficially, this can be achieved without theneed for a sterile environment, without the use of large quantities ofexpensive endotoxin free water and without the need for rinsing orwashing. For example, a low endotoxin alkali chitosan can be reactedwith a greater level of an appropriate acid. A small portion of the acidwill react with the base to make a biocompatible salt.

In another example, low endotoxin alkali chitosan can also be used as araw material for the manufacture of low endotoxin chitosan derivatives,such as carboxy methyl chitosan.

Effect of Acid Salt on Viscosity

Reacting the low endotoxin alkali chitosan with acid, to produce eithera neutral pH chitosan or a chitosan salt, produces an acid saltby-product. The presence of this by-product can affect the performanceof the chitosan product. For example, the level of by-product can affectthe viscosity of a chitosan product in saline.

Referring to FIG. 1 , there is shown the results of adding sodiumlactate to saline in different concentrations, and the resulting effectof this on the viscosity of a 2 g sample of the current market-availablechitosan product, CELOX®, in a 20 g solution of the different mediaafter three minutes.

The base media was saline from body fluids, to which different levels ofsodium lactate were added. The sodium lactate represented the by-productof the reaction between sodium hydroxide and lactic acid.

The results are set out in Table 1 and FIG. 1 .

TABLE 1 Viscosity Concentration Test 1 Test 2 Test 3 Average Saline55000 62000 59000 58667 1.5% 54000 52000 61000 55667 2.5% 50000 4300039000 44000 5.0% 35000 51000 31000 39000 7.5% 35000 34000 38000 3566710.0% 37000 32000 36000 35000

It is clear from FIG. 1 that as the added salt level increases, theviscosity of the CELOX® in the media drops. It is therefore beneficialfor there to be only a low level of residual salt by-product resultingin the chitosan products of the present invention.

Effect of Low Concentration Alkali Solution on Viscosity

The low endotoxin alkali chitosan of the present invention can be testedto demonstrate the effect of the treatment with acid on the viscosity ofthe chitosan polymer, considered to be a measure of molecular weight.The test comprises the following method steps:

-   -   a) weigh out 5 g of low endotoxin alkali chitosan granules;    -   b) weigh out 4.95 g of acetic acid in 600 ml beaker;    -   c) add 490.05 g deionised water to the beaker to make up 495 g        of a 1% solution of acetic acid;    -   d) place the beaker on stirrer plate and turn on stir (increase        as the viscosity of the solution increases;    -   e) add the chitosan granules to the acetic acid solution;    -   f) check the solution regularly until all the granules have        dissolved and increase stirring level as the viscosity of the        solution increases, if required;    -   g) leave the solution for a total of 24 hours, measured from the        time the chitosan granules were introduced into the acetic acid        solution;    -   h) attach a spindle 64 to a Brookfield Viscometer    -   i) set the spindle to 10 rpm;    -   j) insert the spindle into the solution to the mark on the        spindle and turn the viscometer on and allow to stabilise;    -   k) record the viscosity (cPs) at selected time intervals.        Effect of Lowering the Concentration Alkali Solution

The effect of using a lower concentration of alkali solution in theprocess of the present invention can be tested in three experiments,focussing on (1) the percentage penetrability of saline into a testsample; (2) the time period to blood clotting; and (3) the percentagehaemostasis in epigastric sever in-vivo models.

The general test method for (1) the percentage penetrability into salineis as follows: 5 mls of distilled water is added to a test tube. A dropof red food dye is added to the water. 3 g of sample haemostatic powderis gently tipped on top of the water such that a layer is formed. After1 minute, the distance traveled by the water into the haemostatic powderis measured and recorded as percentage penetration.

The general test method for (2) the time period to blood clotting is asfollows: 0.75 g of sample haemostatic powder is added to a test tube, towhich 5 ml of heparinised rabbit blood is added. The test tube is theninverted and the time taken to fully clot the blood into a gel massrecorded.

The general test method for (3) the percentage haemostasis in epigastricsever in-vivo models is as follows: a 3-5 cm sever is made in theepigastric artery of a swine model (non-heparinised). The haemostaticmaterial in granular form is applied and a 1 minute compression applied.If re-bleeding occurs, a further 1 minute compression is undertaken.

It is of course to be understood that the present invention is notintended to be restricted to the foregoing examples which are describedby way of example only.

The invention claimed is:
 1. A process for producing a low endotoxinalkali chitosan, chitin or a derivative thereof having an endotoxinconcentration of less than 50 EU/g, the process comprising the steps of:(a) contacting chitosan, chitin, a chitosan derivative or a chitinderivative with an alkali solution having a concentration of from 0.01Mto 0.2M to form a mixture, wherein the chitosan derivative or chitinderivative is selected from the group consisting of carboxymethylchitosan, hydroxyl butyl chitin, N-acyl chitosan, O-acyl chitosan,N-alkyl chitosan, O-alkyl chitosan, N-alkylidene chitosan, O-sulfonylchitosan, sulfated chitosan, phosphorylated chitosan, nitrated chitosan,alkalichitin, alkalichitosan, and metal chelates with chitosan; and (b)leaving the mixture for a period of less than 12 hours; and then (c)drying the mixture.
 2. The process as claimed in claim 1, wherein theconcentration of the alkali solution is about 0.1M.
 3. The process asclaimed in claim 1, wherein the alkali solution comprises an alkali oralkaline earth component selected from the group consisting of metalhydroxides, metal carbonates, metal bisulphites, metal persilicates,conjugate bases, ammonium hydroxide, and combinations thereof, whereinthe metal is optionally selected from the group consisting of sodium,potassium, calcium, and magnesium, and wherein the alkali component isselected from the group consisting of sodium hydroxide, potassiumhydroxide and sodium carbonate.
 4. The process as claimed in claim 1,wherein the alkali solution is sprayed onto the chitosan, chitin,chitosan derivative or chitin derivative.
 5. The process as claimed inclaim 1, wherein the mixture is left for a period of less than tenhours.
 6. The process as claimed in claim 1, wherein a preservative isadded to the mixture of step (a), wherein the preservative is optionallyselected from silver ions, zinc ions, chlorohexadine, or combinationsthereof.
 7. The process for producing a low endotoxin neutral chitosan,a chitosan salt or a chitosan derivative having an endotoxinconcentration of less than 50 EU/g comprising the step of contacting theproduct of the process of claim 1 with an acid.
 8. The process asclaimed in claim 7, wherein the step of contacting the alkali chitosanwith an acid is performed before a drying step (c); and wherein the acidis optionally sprayed onto the alkali chitosan or the alkali chitosan ismixed with the acid.
 9. The process as claimed in claim 7, wherein theacid is selected from the group consisting of: organic acids optionallyselected from the group consisting of acetic acid, tartaric acid, citricacid, ascorbic acid, acetylsalicylic acid, gluconic acid, lactic acidand combinations thereof; carboxylic acids; fatty acids optionallyselected from the group consisting of myristoleic acid, palmitoleicacid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleicacid, linoelaidic acid, α-Linolenic acid, arachidonic acid,eicosapentaenoic acid, erucic acid, docosahexaenoic acid, caprylic acid,capric acid, lauric acid, myristic acid, palmitic acid, stearic acid,arachidic acid, behenic acid, lignoceric acid, cerotic acid, andcombinations thereof; amino acids optionally selected from the groupconsisting of histidine, lysine, aspartic acid, glutamic acid,glutamine, glycine, proline, taurine, and combinations thereof; lewisacids; monoprotic acids, diprotic acids, and mineral acids; polyproticacids; and nucleic acids.
 10. The process as claimed in claim 7, whereinthe acid has a concentration of about 1M.
 11. The process as claimed inclaim 7, wherein the acid is present as an acid liquor comprising theacid and a non-solvent optionally selected from the group consisting ofethyl lactate, ethyl acetate, methyl acetate, ethanol, acetone, 80:20mixture of ethanol:water, and mixtures thereof.
 12. The process asclaimed in claim 11, wherein the ratio of chitosan to acid liquor isfrom about 5:1 to about 1:5.
 13. The process as claimed in claim 7,further comprising the step of drying the reaction product.
 14. Theprocess as claimed in claim 12, wherein the alkali chitosan is mixedwith the acid for about 5 minutes.
 15. The process as claimed in claim1, wherein the chitosan, chitin, chitosan derivative or chitinderivative is mixed with the alkali solution.
 16. The process as claimedin claim 9, wherein the mineral acid is selected from the groupconsisting of hydrochloric acid, sulphuric acid, nitric acid andcombinations thereof.
 17. The process as claimed in claim 1, whereinneither step of the process involves the use of endotoxin-freeequipment.
 18. The process as claimed in claim 1, wherein the quantityof chitosan, chitin, chitosan derivative or chitin derivative to alkalisolution is from 1:10 to 10:1.
 19. The process as claimed in claim 1,wherein neither step of the process involves a washing step, a rinsingstep, use of a surfactant or phase transfer agents, and/or the use ofendotoxin free water.
 20. The process as recited in claim 1, whereby theviscosity of the low endotoxin alkali chitosan, chitin, or derivativethereof is reduced by less than 25%.